Staphylococcus aureus is a major cause of bacterial infection in the United States, with about half a million infections reported annually. Clinically,S. aureus most often presents as skin/soft tissue infections but it can lead to more severe conditions such as endocarditis, osteomyelitis and sepsis. S. aureus is a successful pathogen due in part to its ability to resist numerous host innate immune effectors including nitric oxide (NO ). S. aureus NO -resistance distinguishes this species from other less pathogenic members of the staphylococci including S. epidermidis and S. saprophyticus. NO generally limits bacterial growth by attacking the active sites of various enzymes thereby constraining the metabolic capabilities of invading pathogens. S. aureus NO - resistance entails the induction of a metabolic state that is resistant to the effects of NO . We are interested in these metabolic adaptations mounted by S. aureus in the face of NO -stress. One enzyme necessary for S. aureus NO -resistance is a lactate:quinone oxidoreductase (Lqo), which is the founder of a family of homologous enzymes only found within the genus Staphylococcus. Lqo allows S. aureus to use available L- lactate as a carbon/energy source in the face of host NO and is therefore required for full virulence. While S. aureus encodes a single Lqo enzyme, other staphylococcal species harbor up to four Lqo homologs. Phylogenetic analyses of these homologs predict that the Lqo family evolved from a similar enzyme (Mqo, oxidizing malate rather than lactate) commonly found among many bacterial species. In Aim 1, I propose to characterize the interaction of Lqo with its substrate through crystallization of the enzyme and mutation of relevant amino acids within the active site. I further describe experiments designed to define the mechanism of substrate specificity among these highly homologous enzymes. Finally, I will screen for small-molecule inhibitors of the Lqo family, compounds that may have therapeutic or research applications. Thus, the Lqo family can be studied as a model of gene duplication/diversification allowing for metabolic flexibility and potentially contributing to speciation and niche adaptation. In Aim 2, I will address how S. aureus incorporated Lqo into its NO -resistant metabolic scheme. Lqo is a respiratory enzyme and requires a terminal electron acceptor for activity. However, NO blocks aerobic respiration by interacting with cytochrome hemes of respiratory terminal oxidases. S. aureus detoxifies NO to nitrate, an anaerobic terminal electron acceptor that might enable Lqo activity under NO -stress. I have found that either the major cytochrome aa3 oxidase or the nitrate reductase can support full NO -resistance. Accordingly, I will determine the relative contributions of both electrons acceptors to NO -survival. Also, I will test whether nitrate reductase, nitrite reductase and nitrite export are involved in S. aureus NO -resistance. In the end, we will have a deeper understanding of the structure, function and evolution of this important family of staphylococcal enzymes. Additionally, we will ascertain how Lqo was incorporated into the S. aureus NO -resistant metabolic scheme thereby contributing to the emergence of a pathogen from a genus of commensal organisms.